ABSTRACT The ?4-allele isoform of apolipoprotein E (ApoE4) plays a key-role in Alzheimer's disease and cardiovascular pathologies. A large body of evidence support that conformations of the protein are instrumental in its contribution to function and disease; yet, much remains unknown about the conformational ensemble of full-length ApoE and its role in protein (dis)function, largely because of its elevated propensity for aggregation/oligomerization and inherent flexibility. In our lab, we have overcome these complications by harnessing state-of-the-art single-molecule fluorescence spectroscopy and, for the first time, we are able to access the structural ensemble of the monomeric full-length ApoE4 (free in solution, embedded in oligomers, and bound to lipids). Our preliminary data clearly indicate that ApoE4 adopts at least three distinct conformers ? previously unidentified ? that coexist in equilibrium. These conformers are highly dynamic and malleable to oligomerization and lipid binding. These novel observations led us to hypothesize that single-point mutations and small molecules can perturb the structural ensemble favoring/disfavoring specific ?pathogenic? conformations, whereas interaction with ligands (e.g. lipids) alters the equilibrium between the different conformers selecting for specific conformations that are required for protein function. Our approach will allow us to determine the conformational properties of ApoE, probe current structural models, and test our hypothesis. The specific aims are as follows: 1) determine the conformational changes and domain movements within the pathogenic ApoE4 and elucidate how point mutations and small molecules modulate protein toxicity; 2) understand the mechanism of lipid interactions, from synthetic lipid vesicles to physiological lipoproteins. A comprehensive description of the structural conformations and dynamics of ApoE in the presence and absence of lipids will shed light on the molecular mechanism behind its role in health and disease, paving the way to developing new therapeutic strategies.